Proposal focuses on the mechanism of HIV assembly, in particular on how the core structural protein Gag traffics within the cell and the host factors involved. We will investigate the relationship between HIV assembly and a putative Gag cofactor, Rab24, a protein involved in autophagy. Rab24 localizes to late endosomes, the site of HIV budding macrophages. Rab proteins are small GTPases involved in vesicle trafficking, some being important for HIV production. Along with rabs, HIV utilizes other components of cellular vesicle machinery for its own assembly and egress. Our preliminary studies show an interaction between Rab24 and HIV Gag, colocalization of Rab24 and Gag within the cell, and that efficient HIV assembly requires Rab24. We hypothesize that HIV uses Rab24 for its assembly/egress, and that Gag trafficking uses autophagic machinery Aim 1: To characterize the molecular basis of interaction between HIV Gag and Rab24. Preliminary studies show that Gag and Rab24 can interact in human cells. We will map the regions of Rab24 and Gag that are important for interaction in human cells. We will also determine if the interaction is direct or indirect, as well as the strength of interaction. Aim 2: To examine the subcellular localization of HIV Gag and Rab24 in physiological target cells of HIV. We have observed colocalization between HIV Gag and Rab24 or a Rab24 mutant in 293T and HeLa cells. We aim to determine the subcellular localization of these proteins in macrophages and CD4+ T-cells by confocal microscopy. To explore the function of Rab24 in assembly, we will also visualize changes in Gag localization that occur upon induction of autophagy. Aim 3: To determine the role of Rab24 and autophagy on HIV production in physiological target cells of HIV. Overexpression of a dominant-active Rab24 mutant increased HIV production. Conversely, knockdown Of Rab24 by siRNA decreased virus production. To determine if Rab24 is important for assembly in physiological targets of HIV infection, we will analyze the role of Rab24 on HIV production in primary macrophages and CD4+ T-cells. Since Rab24 is involved in autophagy, we will determine if autophagy alters HIV production in macrophages and T-cells. Relevance to public health: Currently, there are no drugs targeting HIV assembly. The study of HIV assembly may lead to new therapeutic strategies that are necessary for combating drug resistance and providing alternatives for patients who are refractory to current treatments.